The major established physiologic mechanism for cellular initiation of the human extrinsic coagulation protease cascade is attributed to expression of the glycoprotein Tissue Factor (TF) (Morrisey et al. (1987) Cell 50:129-135) on the surface of cells (Broze (1982) J. Clin. Invest. 70:526-535); Ploplis et al. (1987) J. Biol. Chem. 262:9503-9508). TF is a cell surface receptor that specifically binds plasma factor VII, or its more active two chain derivative VIIa, at a 1:1 ratio to form the binary complex [TF:VII] or [TF:VIIa], hereafter referred to as [TF:VII/VIIa]. The catalytically active moiety is termed herein factor VII/VIIa and possesses esterase activity (Zur and Nemerson (1978) J. Biol. Chem. 253:2203-2209) even in the absence of binding to TF. The [TF:VII/VIIa] binary complex is proteolytically active (Nemerson and Gentry (1986) Biochem. 25:4020-4033) due to substrate association with the functional serine protease type active site of the factor VII/VIIa of the complex. [TF: VII/VIIa] acts as a protease that is highly specific for two other proteins, the serine protease zymogens, factor X (Silverberg et al. (1977) J. Biol. Chem. 252:8481-8488) and factor IX (Osterud et al. (1977) Proc. Natl. Acad. Sci. 74:5260-5264), both of which are in turn rendered active as proteases by the action of the serine type catalytic site of factor VII/VIIa when organized into the bimolecular complex [TF:VII/VIIa]. Due to the specificity of [TF:VII/VIIa] for factor IX, [TF:VII/VIIa] is also capable of activating the intrinsic coagulation protease cascade on some cells, for example on endothelial cells of the vasculature (Stern et al. (1984) Proc. Natl. Acad. Sci. 81:913-917). Initiation of one or both coagulation protease cascades on the surface of intravascular cells is a critically important pathogenetic basis for initiation of thrombosis (Niemetz and Fani (1973) Blood 42:47-59; Stern et al. (1984) supra) and disseminated intravascular coagulation (Niemetz and Fani (1971) Nature New Biol. 232:247-248). Both coagulation protease cascades play a role in the inflammatory response to viruses and immune mediated diseases (Levy et al. (1981) J. Exp. Med. 254:1150-1163).
There are no prior established inhibitors specific for factor VII/VIIa when free or part of the [TF:VII/VIIa] binary complex. There are no known specific inhibitors of this "initiation" of the coagulation protease cascades by the proteolytically active [TF:VII/VIIa] bimolecular complex.
The present invention, providing the ability to specifically inhibit proteolytic activity of the [TF:VII/-VIIa] binary complex, represents a significant and useful advance. Compounds of the invention permit diagnostic evaluation of the molecular basis of cellular activation of coagulation in the thrombotic, inflammatory and related intravascular coagulation and immunologic diseases. Second, such compounds permit analysis for the development of drugs based on the activity of the [TF:VII/VIIa] complex. Third, such compounds represent a new class of anti-thrombotic and anti-inflammatory drugs.
The usefulness of the class of compounds described in this invention derives from the ability of these compounds to function a active site inhibitors specific for factor VII/VIIa. Both reversible and irreversible inhibitors are described. The irreversible inhibitors are capable of efficient inhibition and are selective for factor VII/VIIa when active by association with tissue factor (TF)in the proteolytically active binary complex [TF:VII/VIIa].
The compounds of the invention are used as analytical reagents and therapeutic agents to specifically inhibit the initiation of the coagulation protease cascades by [TF:VII/VIIa]. The compounds also permit accurate in vitro and ex vivo determination whether or not activation of coagulation is attributable to the binary complex [TF:VII/VIIa]. The compounds are used as therapeutic drugs in vivo to inhibit the initiation of the coagulation system which is one of the pathogenetic mechanisms required for thrombus formation and the thrombotic and related diseases, disseminated intravascular coagulation associated with septic shock and other disease processes, and certain inflammatory conditions associated with excessive activation of coagulation in the tissues.